Liquid chromatography–tandem mass spectrometry determination of LSD, ISO-LSD, and the main metabolite 2-oxo-3-hydroxy-LSD in forensic samples and application in a forensic case

Abstract

A liquid chromatography mass spectrometric (LC/MS/MS) method has been developed for the determination of LSD, iso-LSD and the metabolite 2-oxo-3-hydroxy-LSD in forensic applications. The procedure involves liquid–liquid extraction of the analytes and LSD-D₃ (internal standard) from 1.0 g whole blood or 1.0 ml urine with butyl acetate at pH 9.8. Confirmation and quantification were done by positive electrospray ionisation with a triple quadrupole mass spectrometer operating in multiple reaction monitoring (MRM) mode. Two MRM transitions of each compound were established and identification criteria were set up based on the retention time and the ion ratio. The curves of extracted standards were linear over a working range of 0.01–50 μg/kg for all transitions of LSD and iso-LSD. The limit of quantification was 0.01 μg/kg for LSD and iso-LSD. The method was applied to a case investigation involving a 26-year-old male suspected for having attempted homicide, where blood concentrations of LSD and iso-LSD were determined to 0.27 and 0.44 μg/kg, respectively. 2-Oxo-3-hydroxy-LSD was detected in the urine and confirmed the LSD abuse. The case illustrated the importance of analyte separation before MRM detection of a sample due to identical fragmentation ions of the isomers.

Introduction

Lysergic acid diethylamide (LSD) illustrated in Fig. 1 is a very potent hallucinogenic substance. It is considered a highly dangerous drug due to its tendency to produce panic, delirium and bizarre behaviour, sometimes resulting in irrational and injurious acts [1], [2], [3], [4]. LSD acts on multiple sites at the CNS. Its best studied effects are the agonistic actions at presynaptic receptors for 5-hydroxy-tryptamine in the midbrain [1], [5]. LSD is an illegal drug commonly referred to as “acid” and it does not occur in nature but is easily prepared from ergotamine and related ergot alkaloids [6]. LSD is colourless, odourless and tasteless and primarily found on blotter paper [1], [2], [3], [4], [5]. The LSD consumption has increased since the beginning of the 1990s after a drop in the 1980s. The active oral dose is about 20–100 μg, but sometimes up to 500 μg is used [1], [2], [3], [4], [7]. The active blood level concentration of LSD is very low (maximum plasma concentrations following 70 μg dose range up to 2 μg/kg). The plasma elimination half-life of LSD has been reported to be 3–6 h [1], [2], [3], [4], [5]. LSD is extensively metabolized in the liver and less than 1% of the drug is eliminated unchanged in the urine [1], [2], [3], [4]. Because of the low blood concentrations, LSD analysis is often performed on urine. Nor-LSD, 2-oxo-3-hydroxy-LSD and the glucuronide conjugate of 13-hydroxy-LSD can be detected in urine up to 96 h after administration, whereas LSD can only be detected for 12–24 h post administration. 2-Oxo-3-hydroxy-LSD is present in human urine at concentrations 16–43 times greater than LSD, the parent compound [8]. Iso-LSD (an inactive isomer at C-8) is often found in urine, but it is a contaminant of LSD itself.

As a result of the high potency and low doses, detection of LSD in biological matrices poses a significant analytical challenge. Direct GC/MS is of limited use because of the irreversible adsorption of LSD on GC columns and because of its low volatility and thermal instability at GC temperatures. Selective extraction and derivatization by silylation of the indole nitrogen of LSD is necessary when using GC/MS. Alternatively HPLC separation together with fluorescence detection has been applied, however, it lacks sensitivity and specificity [7], [8], [9], [10], [11], [12]. Consequently, LC/MS/MS is studied as the method of choice for LSD determination in biological samples.

Most publications describe methods for urine due to the high sensitivity demands [10], [11], [12]. Only few papers have described analytical methods sensitive enough for determination of LSD and related analytes in blood [13], [14]. Faverretto et al. [13] described a simple LC/MS/MS using step liquid–liquid extraction of biological samples with the carcinogenic solvent chloroform, while Canezin et al. [14] describe a LC/MS/MS method for plasma and urine with a similar one step liquid–liquid extraction using diethylether (extremely explosive). Both methods involved hazardous solvents but described sufficient sensitivity with acceptable sample size. Other more complicated methods involving difficult and time-consuming sample preparation are described such as a LC/MS method by Bodin and Svensson [15] involving extraction into an organic solvent (diisopropylether) and back-extraction. Sklerov et al. [16] reported a LC/MS method for whole blood of a combined liquid–liquid extraction and solid-phase extraction. Many earlier methods lack the required sensitivity for measuring LSD in blood such as Bogusz et al. [17], who used LC–APCI–MS for routine in multipleanalyte analysis. Use of tandem mass spectrometry clearly improves and simplify the sample preparation.

The Department of Forensic Chemistry at the Institute of Forensic Medicine performs the toxicological analysis on the driving and criminal cases (offences) in Denmark and the post-mortem cases of East Denmark requested by the police. This paper describes a LC/MS/MS method using positive ESI with a triple quadrupole mass spectrometer for the determination of LSD and related analytes in biological samples from forensic cases. A case is studied involving a 26-year-old male suspected of having attempted homicide. The method describes a new simple sample preparation method of forensic samples involving one-step liquid–liquid extraction using a more environmental safe/less hazardous solvent than earlier described methods.